Biology subjects

Schuller, M.

Publications and source records attributed to Schuller, M..

5 recordsLinked to original sources

bioRxiv2026

DarHTG is a tripartite phage defence system for purine DNA base ADP-ribosylation

Bacteria evolved a variety of phage defence strategies to combat the constant onslaught of viral threats. DarTG toxin-antitoxin systems provide phage defence by ADP-ribosylation of bases in single-stranded DNA which interferes with viral replication and aborts phage infection. Here, we describe DarHTG, a system widespread among bacteria, that combines DarTG with a non-processive superfamily I helicase functioning as co-toxin for DarT and thereby enabling the ADP-ribosylation of double-stranded DNA. We identified two such DarHTG subclasses which catalyse the ADP-ribosylation of adenosine and guanosine bases, respectively, and reverse these modifications with structurally unique ARH superfamily antitoxins. Thus, DarHTG antitoxins expand the known macromolecular target spectrum of the ARH enzyme class to nucleic acids. These DarHTG systems confer resistance to a broad range of bacteriophages and can be triggered by the highly charged tails of viral single-stranded DNA-binding proteins, which occlude the active site of the ARH antitoxin.

microbiology↗

bioRxiv2023

PARP14 is a PARP with both ADP-ribosyl transferase and hydrolase activities

PARP14 is a mono-ADP-ribosyl transferase involved in the control of immunity, transcription and DNA replication stress management. However, little is known about the ADP-ribosylation activity of PARP14, including its substrate specificity or how PARP14-dependent ADP-ribosylation is reversed. Here we show that PARP14 is dual function enzyme with both ADP-ribosyl transferase and hydrolase activity acting on both protein and nucleic acid substrates. In particular, we show that the PARP14 macrodomain 1 is an active ADP-ribosyl hydrolase. We also demonstrate hydrolytic activity for the first macrodomain of PARP9. We reveal that expression of a PARP14 mutant with the inactivated macrodomain 1 results in a dramatic increase in mono(ADP-ribosyl)ation of proteins in human cells, including PARP14 itself and antiviral PARP13. Moreover, we demonstrate that the closely related hydrolytically active macrodomain of SARS2 Nsp3, Mac1, efficiently reverses PARP14 ADP-ribosylation in vitro and in cells, supporting the evolution of viral macrodomains to counteract PARP14-mediated antiviral response. TeaserPARP14 is an antiviral PARP that combines ADP-ribosylation writer, reader and eraser functions in one polypeptide.

cell biology↗

bioRxiv2022

Fragment-Based Hit Discovery via Unsupervised Learning of Fragment-Protein Complexes

The process of finding molecules that bind to a target protein is a challenging first step in drug discovery. Crystallographic fragment screening is a strategy based on elucidating binding modes of small polar compounds and then building potency by expanding or merging them. Recent advances in high-throughput crystallography enable screening of large fragment libraries, reading out dense ensembles of fragments spanning the binding site. However, fragments typically have low affinity thus the road to potency is often long and fraught with false starts. Here, we take advantage of high-throughput crystallography to reframe fragment-based hit discovery as a denoising problem - identifying significant pharmacophore distributions from a fragment ensemble amid noise due to weak binders - and employ an unsupervised machine learning method to tackle this problem. Our method screens potential molecules by evaluating whether they recapitulate those fragment-derived pharmacophore distributions. We retrospectively validated our approach on an open science campaign against SARS-CoV-2 main protease (Mpro), showing that our method can distinguish active compounds from inactive ones using only structural data of fragment-protein complexes, without any activity data. Further, we prospectively found novel hits for Mpro and the Mac1 domain of SARS-CoV-2 non-structural protein 3. More broadly, our results demonstrate how unsupervised machine learning helps interpret high throughput crystallography data to rapidly discover of potent chemical modulators of protein function.

bioinformatics↗

bioRxiv2022

Structure-based inhibitor optimization for the Nsp3 Macrodomain of SARS-CoV-2

The nonstructural protein 3 (NSP3) of the severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) contains a conserved macrodomain enzyme (Mac1) that is critical for pathogenesis and lethality. While small molecule inhibitors of Mac1 have great therapeutic potential, at the outset of the COVID-19 pandemic there were no well-validated inhibitors for this protein nor, indeed, the macrodomain enzyme family, making this target a pharmacological orphan. Here, we report the structure-based discovery and development of several different chemical scaffolds exhibiting low- to sub-micromolar affinity for Mac1 through iterations of computer-aided design, structural characterization by ultra-high resolution protein crystallography, and binding evaluation. Potent scaffolds were designed with in silico fragment linkage and by ultra-large library docking of over 450 million molecules. Both techniques leverage the computational exploration of tangible chemical space and are applicable to other pharmacological orphans. Overall, 160 ligands in 119 different scaffolds were discovered, and 152 Mac1-ligand complex crystal structures were determined, typically to 1 [A] resolution or better. Our analyses discovered selective and cell-permeable molecules, unexpected ligand-mediated protein dynamics within the active site, and key inhibitor motifs that will template future drug development against Mac1. Significance StatementSARS-CoV-2 encodes a viral macrodomain protein (Mac1) that hydrolyzes ribo-adenylate marks on viral proteins, disrupting the innate immune response to the virus. Catalytic mutations in the enzyme make the related SARS-1 virus less pathogenic and non-lethal in animals, suggesting that Mac1 will be a good antiviral target. However, no potent inhibitors of this protein class have been described, and pharmacologically the enzyme remains an orphan. Here, we computationally designed potent inhibitors of Mac1, determining 150 inhibitor-enzyme structures to ultra-high resolution by crystallography. In silico fragment linking and molecular docking of > 450 million virtual compounds led to inhibitors with submicromolar activity. These molecules may template future drug discovery efforts against this crucial but understudied viral target.

biophysics↗

bioRxiv2020

Fragment binding to the Nsp3 macrodomain of SARS-CoV-2 identified through crystallographic screening and computational docking

The SARS-CoV-2 macrodomain (Mac1) within the non-structural protein 3 (Nsp3) counteracts host-mediated antiviral ADP-ribosylation signalling. This enzyme is a promising antiviral target because catalytic mutations render viruses non-pathogenic. Here, we report a massive crystallographic screening and computational docking effort, identifying new chemical matter primarily targeting the active site of the macrodomain. Crystallographic screening of diverse fragment libraries resulted in 214 unique macrodomain-binding fragments, out of 2,683 screened. An additional 60 molecules were selected from docking over 20 million fragments, of which 20 were crystallographically confirmed. X-ray data collection to ultra-high resolution and at physiological temperature enabled assessment of the conformational heterogeneity around the active site. Several crystallographic and docking fragment hits were validated for solution binding using three biophysical techniques (DSF, HTRF, ITC). Overall, the 234 fragment structures presented explore a wide range of chemotypes and provide starting points for development of potent SARS-CoV-2 macrodomain inhibitors.

biochemistry↗